Biopharmaceutical products from eukaryotic cells are an integral part of modern medicine. However, increased productivity and increased safety are important parameters that still require substantial optimization. The most crucial bottleneck in the efforts towards optimization is the cellular substrate itself. A safe transgene that can be introduced into cell lines already released for use in biopharmaceutical processes to increase product yields from the such manipulated cells is extremely valuable.
Adenoviruses are unenveloped (naked) double-stranded DNA viruses that infect a broad spectrum of animals. Among the best characterized members is the human adenovirus serotype 5 (Ad5). This virus is a frequent cause of common cold symptoms and infection often occurs in childhood.
Adenoviruses are amenable to genetic manipulation and replication incompetent adenoviruses, including Ad5 (GenBank accession number for sequence: AC—000008), are used as vectors in gene therapy and for therapeutic vaccination. To obtain replication incompetent viruses large regions of the genomic DNA are substituted with non-viral sequences. The missing viral functions are provided in trans by host cell lines that have been stably transfected with genes that are deleted in the vector. One of the earlier systems that were developed consist of adenoviral vectors deleted in the regulatory E1 region produced on cell lines providing the corresponding E1 proteins.
The most common cell line for this purpose is the 293 cell line. This line was generated in 1977 by transfection of fragmented adenoviral genomic DNA into primary human cells (Graham et al., J. Gen. Virol. 36, 59-74 (1977)), well before it was recognized that adenoviruses may serve as gene therapy vectors. The resulting cell line was subsequently shown to contain nucleotides 1 to 4344 of the genomic DNA (Louis et al., Virology 233, 423-429 (1997)) which includes the E1 region; this characterization demonstrated that the E1 region can be used to immortalize and transform primary cells.
Adjacent to the E1 region follows the gene for pIX (“protein 9”; nucleotides 3609 to 4031 on the genomic DNA). The promoter for pIX is embedded within the E1B component of the E1 region. A mechanism called promoter occlusion allows expression of pIX at a delayed-early time in the viral infection cycle at the onset of viral DNA replication with increasing copy number of viral DNA (Fessler and Young, J. Virol. 72, 4049-4056 (1998)). Although the gene is present within the 4344 nucleotides integrated into 293 cells, pIX expression cannot be detected even with sensitive radioactive labeling methods (Spector et al., J. Virol. 36, 860-871 (1980)).
As described above, replication deficient adenoviral vectors have been generated by deletion of the E1 region from the viral genome. E1 products are essential for viral replication and the function of the E1 region therefore had to be provided in trans via stable E1 transgenes in the adenovirus packaging cell (such as the 293 cell line). Because of the proximity to the E1 region the pIX gene caused frequent recombination between deleted adenoviral vectors and E1 in the host genome. This recombination event generated replication competent adenovirus (RCA), a serious contamination in vector preparations. To suppress this recombination event pIX was deleted from the adenovirus genome. In the course of these experiments it was recognized that pIX stabilizes the adenoviral capsid against thermal and steric stress by increasing the interaction of the main building blocks, the hexons (Colby and Shenk, J. Virol. 39, 977-980 (1981); Ghosh-Choudhury et al., EMBO J. 6, 1733-1739 (1987)). Morphogenesis in the absence of pIX yields temperature-sensitive viruses that cannot deliver genomic DNA molecules larger than 105% of the wild type 35938 base pairs. To still allow a normal packaging capacity and thermal stability the pIX protein was stably introduced into cell lines intended as packaging cells for adenovirus vectors (Krougliak and Graham, Hum. Gene Ther. 6, 1575-1586 (1995); WO 99/57296 and Imler et al., Gene Therapy 3, 75-84 (1996)).
Also with recognition that pIX decorates the surface of virions pIX-fusion proteins have been generated with the purpose to expand the host range of adenovirus vectors or to follow morphogenesis and the intracellular migration of virus particles (reviewed by Parks, Mol. Ther. 11, 19-25 (2005)).
Although clearly a structural protein, pIX also is implicated as regulatory protein for adenovirus replication. PIX is assumed to function as transactivator of transcription to augment E1A expression, a function possibly even excerted as a virokine by incoming PIX protein from the capsid at the infection step (reviewed by Parks, Mol. Ther. 11, 19-25 (2005)). PIX, together with early protein E4 Orf3, also is described to interact with sub-nuclear inclusions called PML bodies (Puvion-Dutilleul et al., Exp. Cell. Res. 218, 9-16 (1995); Leppard and Everett, J. Gen. Virol. 80, 997-1008 (1999)). These are dynamic aggregates of 250 nm to 500 nm in size and proposed to be involved in regulation of cell differentiation (Wang et al., Science 279, 1547-1551 (1998)), control of apoptosis (Quignon et al., Nature Gen. 20, 259-265 (1998)), and response to viral infection (Möller and Schmitz, Arch Immunol Ther Exp (Warsz) 51, 295-300 (2003)).
Because of its pleotropic effects we introduced the PIX protein into cell lines to examine whether PIX augments cell proliferation or production properties for biopharmaceutical products that are not related to adenovirus or adenoviral vectors. There is a general need for factors that modulate these properties in established cell lines.
For example, attenuated (weakened) viruses are promising vaccine candidates: upon inoculation they mimic a natural infection but allow more time for establishment of the desired protective immune response by the vaccinee. With increasing numbers of immunocompromised patients (for example, due to HIV infection) highly attenuated strains are desirable. Whereas attenuated strains still proceed towards (usually benign) infection highly attenuated strains are blocked at a cellular level even in absence of a functional immune system. A frequently used method to establish and maintain attenuation is to passage viruses on different host tissue. For example, measles and mumps viruses intended for human vaccination are passaged in primary cells, either in embryonated chicken eggs or cultures derived thereof. A new generation of vaccines is based on highly attenuated pox viruses that also depend on primary chicken cells for production. A cell line that can substitute for primary chicken cells and at the same time that even less efficiently protects itself against viral infection due to secondary manipulations such as introduction of the PIX transgene therefore provides a highly desirable substrate.
For other purposes a mammalian (rather than avian) cell line may be preferred. Such preferred cell lines already exist and have passed the examination by health authorities with respect to safety and risks posed by derived biopharmaceutical products. Here too, a secondary manipulation (such as introduction of the PIX transgene) to increase the spectrum of available applications or production efficiencies without compromising safety features is highly desirable.